Kaposi's sarcoma-associated herpesvirus (KSHV) is the responsible agent for Kaposi's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV expresses multiple microRNAs that modulate host gene expression. Most microRNAs (miRNAs) repress target gene expression by destabilizing the mRNA transcript and decreasing translational efficiency. A goal of the project is to determine targets of viral miRNAs and understand why the virus has selected specific human target genes for inhibition. We hope to discover new functions of human genes as they relate to viral infection and cancer. Using a variety of expression profiling data, we constructed a dataset to integrate the expression data from multiple gain and loss of microRNA function experiments. We have tested over fifty predicted target genes and over thirty microRNA target genes were significantly inhibited by viral miRNAs using a variety of validation methods. In addition, we identified multiple examples of individual target genes being inhibited by multiple KSHV miRNAs. It is noteworthy to state approximately half of these microRNA:target interactions are not detected using common bioinformatic methods. A subset of these target genes has been further validated by looking at protein expression of endogenous target genes in response to viral microRNA expression, microRNA inhibition in infected cells and KSHV infection. To assess changes in protein expression, we utilize a near-infrared scanner to perform simultaneous two-color quantitative western blotting assays. In addition, we have mapped functional microRNA target sites in multiple human genes using site-directed mutagenesis. Furthermore, using KS biopsies we have determined multiple microRNA target genes that are inhibited in our cell culture systems are also inhibited at sites of KSHV infection in patients. We have also completed a proteomic screen for miRNA targets, which identified novel validated and previously reported miRNA targets. These discoveries have led to novel discoveries about immune evasion and transcription factor regulation. Our analysis found that KSHV microRNAs can inhibit activation of intercellular adhesion molecule 1 (ICAM1) protein expression, by inhibiting rho-associated, coiled-coil containing protein kinase 2 (ROCK2) and signal transducer and activator of transcription 3 (STAT3) activation pathways of ICAM1. Analysis of the proteins that were upregulated in the presence of KSHV microRNAs, led to the discovery that KSHV microRNAs can upregulate hypoxia-inducible factor 1 alpha and heme oxygenase 1. Previous microarray and proteomic studies predicted that multiple splice variants of the tumor suppressor protein tropomyosin 1 (TPM1) were targets of KSHV microRNAs. We showed that at least two microRNAs of KSHV, miR-K2 and miR-K5, repress protein levels of specific isoforms of TPM1. We identified a functional miR-K5 binding site in the 3' untranslated region (UTR) of one TPM1 isoform. Furthermore, the inhibition or loss of miR-K2 or miR-K5 restores expression of TPM1 in KSHV-infected cells. TPM1 protein levels were also repressed in KSHV-infected clinical samples compared to uninfected samples. Functionally, miR-K2 increases viability of unanchored human umbilical vein endothelial cells by inhibiting anoikis (apoptosis after cell detachment), enhances tube formation of HUVECs, and enhances VEGFA expression. Taken together, KSHV miR-K2 and miR-K5 may facilitate KSHV pathogenesis. Finally, we are using network analysis to understand how newly identified microRNA target genes could be interacting with each other. This analysis has revealed multiple examples of how microRNA target genes are in the same signaling pathway. These examples highlight significant pathways targeted by KSHV miRNAs and focusing our efforts on pathways with multiple miRNA targets.